Downregulation of GSK3B by miR-132-3p Enhances Etoposide-Induced Breast Cancer Cell Apoptosis.

OBJECTIVE: MicroRNAs (miRNAs) have been confirmed to play an essential role in modulating cancer cell proliferation, metastasis, and sensitivity to chemotherapy. However, the correlation between miR-132-3p expression and etoposide (VP16) induced apoptosis in human breast cancer cells remains poorly understood. METHODS: Six datasets, including three gene expression profile datasets and three microRNA (miRNA) expression profile datasets, were downloaded from the NCBI Gene Expression Omnibus (GEO) database to identify miR-132-3p and GSK3B expression in breast cancer. Kaplan-Meier and log-rank testing were performed to evaluate the effect of miR-132-3p and GSK3B on the survival of breast cancer. Flow cytometry analysis was used to determine the effects of miR-132-3p and GSK3B on breast cancer cell apoptosis. Luciferase reporter assay, Western blot, and real-time PCR were used to determine the regulatory effect of miR-132-3p on GSK3B. RESULTS: miR-132-3p was significantly downregulated in breast cancer tissues compared with normal breast epithelial cells, whereas GSK3B expression was remarkably over-expressed in breast cancer tissues. The patients with low miR-132-3p or high GSK3B expression had worse overall survival. Luciferase reporter assay, Western blot, and real-time PCR confirmed that miR-132-3p could inhibit GSK3B protein and mRNA expression via binding to the 3'-UTR of GSK3B. Furthermore, miR-132-3p enhanced VP16-induced breast cancer cell apoptosis through targeting GSK3B. CONCLUSION: Collectively, the results of this study indicated that miR-132-3p was downregulated in breast cancer tissues and directly targeted GSK3B to be implicated in the modulation of breast cancer cell apoptosis, suggesting that miR-132-3p/GSK3B might be a novel, effective therapeutic target for treating patients with breast cancer.

Systematic Review and Pharmacological Considerations for Chloroquine and Its Analogs in the Treatment for COVID-19.

COVID-19 has been announced pandemic by WHO and over 17,000,000 people infected (Till April 21st 2020). The disease is currently under control in China, with a curative rate of 86.8%. Chloroquine (CQ) is an old anti-malarial drug with good tolerability, which had proved to be effective in previous SARS-coronavirus, which spread and disappeared between 2002-2003. In vitro studies demonstrated the efficacy of CQ in curing COVID-19. Consequently, via analytical PBPK modeling, a further preliminary clinical trial has proved the efficacy and safety of CQ in China., and multiple clinical trials were registered and approved to investigate the activity of other analogs of CQ against COVID-19. We have listed all the clinical trials and made a meta-analysis of published data of hydroxychloroquine (HCQ). HCQ could increase the CT improvement and adverse reactions (ADRs) significantly though there was considerable heterogeneity among current researches. Actually, CQ and its analogs have unique pharmacokinetic characteristics, which would induce severe side effects in some circumstances. We have then summarized pharmacological considerations for these drugs so as to provide to the busy clinicians to avoid potential side effects when administered CQ or its analogs to COVID-19 patients, especially in the elderly, pediatrics, and pregnancies.

Genetic biomarkers of drug resistance: A compass of prognosis and targeted therapy in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a highly aggressive hematological malignancy with complex heterogenous genetic and biological nature. Thus, prognostic prediction and targeted therapies might contribute to better chemotherapeutic response. However, the emergence of multidrug resistance (MDR) markedly impedes chemotherapeutic efficacy and dictates poor prognosis. Therefore, prior evaluation of chemoresistance is of great importance in therapeutic decision making and prognosis. In recent years, preclinical studies on chemoresistance have unveiled a compendium of underlying molecular basis, which facilitated the development of targetable small molecules. Furthermore, routing genomic sequencing has identified various genomic aberrations driving cellular response during the course of therapeutic treatment through adaptive mechanisms of drug resistance, some of which serve as prognostic biomarkers in risk stratification. However, the underlying mechanisms of MDR have challenged the certainty of the prognostic significance of some mutations. This review aims to provide a comprehensive understanding of the role of MDR in therapeutic decision making and prognostic prediction in AML. We present an updated genetic landscape of the predominant mechanisms of drug resistance with novel targeted therapies and potential prognostic biomarkers from preclinical and clinical chemoresistance studies in AML. We particularly highlight the unfolded protein response (UPR) that has emerged as a critical regulatory pathway in chemoresistance of AML with promising therapeutic horizon. Futhermore, we outline the most prevalent mutations associated with mechanisms of chemoresistance and delineate the future directions to improve the current prognostic tools. The molecular analysis of chemoresistance integrated with genetic profiling will facilitate decision making towards personalized prognostic prediction and enhanced therapeutic efficacy.

Potential False-Negative Nucleic Acid Testing Results for Severe Acute Respiratory Syndrome Coronavirus 2 from Thermal Inactivation of Samples with Low Viral Loads.

BACKGROUND: Coronavirus disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and management of COVID-19. In some circumstances, thermal inactivation at 56°C has been recommended to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests. METHODS: We investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2, particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times, and a chemical inactivation approach on NAT. RESULTS: Our study showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation. CONCLUSION: Thermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.

Severe Anaphylaxis During Allogeneic Hematopoietic Stem Cell Transplantation in a Patient with Aplastic Anemia: Case Report of Individualized Pharmaceutical Care and Literature Review.

BACKGROUND: Cyclosporine injection is usually applied in allogeneic hematopoietic stem cell transplantation (Allo-HSCT) during induction phase. Anaphylaxis to cyclosporine injection is rare and how to deal with this issue in clinical practice is intractable. METHODS: We report a Chinese male patient with aplastic anemia who underwent allogeneic bone marrow transplantation (BMT) from his brother where HLA totally matched (10/10). Cyclosporine at a dose of 3 mg/kg was started by continuous infusion over 24 hours on day -1 of BMT and the patient showed sever anaphylaxis symptoms. He was then given oral capsules of cyclosporine (Sandimmun) at a conversion ratio 2:1. No further anaphylactic reaction was observed. The BM cells were successfully engrafted without causing severe GVHD. Moreover, frequent TDM monitoring as well as CYP3A4/CYP3A5/MDR1 genotyping were given so as to tailor the oral dosage of cyclosporine individually and prevent the adverse reaction between cyclosporine and posaconazole. RESULTS: The patient carried CYP3A5*3 GG genotype and the concentration of cyclosporine remained steady in the period of conversion and combination of cyclosporine and posaconazole. Consequently, the patient reported no allergy after conversion to oral cyclosporine. CONCLUSIONS: Polyoxyethylated castor oil that is contained in cyclosporine may be the main allergen. Changing to oral capsules that do not contain this medicinal excipient instead of cyclosporine injection would no longer cause an allergic reaction. Rational use of immunosuppressants and prophylaxis antibiotics may need close cooperation between physicians and pharmacists to avoid side effects and harmful interactions.

MiR-129-5p sensitization of lung cancer cells to etoposide-induced apoptosis by reducing YWHAB.

Background: Lung cancer is the most common cause of death from cancer worldwide and recent studies have revealed that microRNAs play critical roles to regulate lung carcinogenesis. microRNA-129-5p (miR-129-5p) has been reported to regulate cell proliferation and invasion in lung cancer, but its role in lung cancer apoptosis remains unknown. Methods: The expression of miR-129-5p and YWHAB in lung cancer tissues were analyzed from data downloaded from the NCBI Gene Expression Omnibus (GEO) database. Luciferase reporter assay, Western blot and qRT-PCR were used to determine the regulatory effect of miR-129-5p on YWHAB. Cell apoptosis was detected by using the PI/Annexin V Cell Apoptosis Kit. The effect of miR-129-5p and YWHAB on the survival of lung cancer patients was also explored. Results: In this study, by combining the data derived from six GEO database, our results showed that miR-129-5p was downregulated in lung cancer tissues and YWHAB was upregulated in lung cancer patient' serum. A significant negative correlation between miR-129-5p and YWHAB was found in lung cancer tissues. Both the expression of YWHAB and miR-129-5p were associated significantly with prognosis (overall survival) in patients with lung cancer. Overexpression of miR-129-5p promotes VP16-induced lung cancer cell apoptosis and YWHAB was shown to be a direct downstream target of miR-129-5p. Conclusion: Overexpression of expression miR-129-5p contributes to etoposide-induced lung cancer apoptosis by modulating YWHAB.

Prognostic significance of osteopontin in acute myeloid leukemia: A meta-analysis.

Osteopontin (OPN) has been investigated in the field of tumor research for several years. However, the prognostic role of OPN overexpression in acute myeloid leukemia (AML) remains controversial. A meta-analysis of four studies, including a total of 492 patients, was performed to determine the association of OPN with overall survival (OS) in AML patients. The random-effects model of Der Simonian and Laird was used to synthesize data; hazard ratio (HR) with its 95% confidence interval (CI) was used as the effect size estimate. It was observed that serum-based OPN was inversely correlated with OS and the difference was statistically significant (HR=1.83; 95% CI: 1.43-2.35; P<0.001). Experimental findings indicate that OPN overexpression is associated with a poor prognosis in AML and may be of prognostic value for AML stage and metastasis.

Comparison of the Target Values Established Using a Robust Statistical Method and Roche's Calibration Values.

BACKGROUND: In EQA schemes, results are usually assessed against a target value. METHODS: Outliers in each result were deleted using a robust statistical method. Mean was used as a target value if trimmed results were normally distributed; otherwise, median was used. Differences between target value and Roche's calibration value were calculated. Rates of acceptable performance evaluated by target value and Roche's calibration value were determined. RESULTS: Target values of P, GLU, urea, UA, TP, ALB, TC, TG, ALT, AST, ALP, AMY, LDH, GGT, FE, and HBDH were 1.70 mmol/L, 11.0 mmol/L, 16.8 mmol/L, 307 µmol/L, 54.0 g/L, 38.1 g/L, 4.19 mmol/L, 1.46 mmol/L, 97.7 U/L, 98.2 U/L, 206 U/L, 180 U/L, 242 U/L, 115 U/L, 35.6 µmol/L, and 255 U/L, respectively. Differences between target values and Roche's calibration values were -3.45 to 1.48%. Rates of acceptable performance evaluated by target values and Roche's calibration values were 96.5 - 100.0% and 93.0 - 100.0%, respectively. CONCLUSIONS: Target values established using a robust statistical method and Roche's calibration values were comparable.

The RNA/DNA-binding protein PSF relocates to cell membrane and contributes cells' sensitivity to antitumor drug, doxorubicin.

Cell surface proteins play an important role in multidrug resistance (MDR). However, the identification involving chemoresistant features for cell surface proteins is a challenge. To identify potential cell membrane markers in hematologic cancer MDR, we used a cell- and antibody-based strategy of subtractive immunization coupled with cell surface comparative screening of leukemia cell lines from sensitive HL60 and resistant HL60/DOX cells. Fifty one antibodies that recognized the cell surface proteins expressed differently between the two cell lines were generated. One of them, the McAb-5D12 not only recognizes its antigen but also block its function. Comparative analysis of immunofluorescence, flow cytometry, and mass spectrum analysis validated that the membrane antigen of McAb-5D12 is a nucleoprotein-polypyrimidine tract binding protein associated splicing factor, PSF. Our results identified that PSF overexpressed on the membrane of sensitive cells compared with resistant cells and its relocation from the nuclear to the cell surface was common in hematological malignancy cell lines and marrow of leukemia patients. Furthermore, we found that cell surface PSF contributed to cell sensitivity by inhibiting cell proliferation. The results represent a novel and potentially useful biomarker for MDR prediction. The strategy enables the correlation of expression levels and functions of cell surface protein with some cell-drug response traits by using antibodies.

PHII-7 inhibits cell growth and induces apoptosis in leukemia cell line K562 as well as its MDR- counterpart K562/A02 through producing reactive oxygen species.

Multidrug resistance (MDR) is a major obstacle that hinders the efficacy of chemotherapy in many human malignancies. PHII-7 is a derivative of indirubin, which was designed and synthesized by our laboratory. Our preliminary work indicated its potent antitumor activities in vitro and in vivo. Furthermore, based on the model of MDR cell line, we found its powerful effects in inhibiting the expression of P-glycoprotein (P-gp) and killing multidrug-resistant (MDR) cells with the detailed mechanism remained to be explored. Reactive oxygen species are known for high reactive activity as they possess unmatched electrons. In this study, we showed that PHII-7 generated equal reactive oxygen species in parental K562 and its counterpart MDR K562/A02 cells. Pre-incubation with thiol antioxidants glutathione or N-acetyl-cysteine(NAC) almost abolished the cytotoxicity of PHII-7. Moreover, NAC abrogated DNA damage, cell cycle arrests and apoptosis induced by PHII-7. Our results collectively indicated that reactive oxygen species production induced by PHII-7 contributed to both apoptosis and cell cycle arrets in MDR K562/A02 cells, thus extending our prior related findings. Notably, JNK phosphorylation was also induced by PHII-7 and pre-incubated of K562/A02 cells with NAC or inhibitor of JNK(SP006125) eliminated P-gp downregulation. Taken together, our results may provide a detailed biochemical basis for further clinical application of PHII-7.

A novel calmodulin antagonist O-(4-ethoxyl-butyl)-berbamine overcomes multidrug resistance in drug-resistant MCF-7/ADR breast carcinoma cells.

Multidrug resistance (MDR) mediated by the overexpression of the drug efflux protein P-glycoprotein is one of the major obstacles to successful cancer chemotherapy. The development of safe and effective MDR-reversing agents is an important approach to addressing this problem clinically. In this study, we evaluated the P-gp-modulatory potential of O-(4-ethoxyl-butyl)-berbamine (EBB), a novel calmodulin antagonist and derivative of bisbenzylisoquinoline alkaloid, which significantly improved the chemosensitivity of P-glycoprotein-mediated multidrug-resistant cells to doxorubicin compared with the efficacy of a conventional P-glycoprotein inhibitor, verapamil. EBB not only blocked the function of P-glycoprotein confirmed by the fact that EBB increased intracellular accumulation of rhodamine 123 and doxorubicin but also inhibited the expression of P-glycoprotein actualized by downregulating P-glycoprotein. Furthermore, our results showed that cotreatment with EBB and doxorubicin resulted in marked G(2)/M arrest and apoptosis of MCF-7/ADR cells, accompanied by down-regulation of the proteins cdc2/p34 and cyclin B1 and increased the levels of calcium ions. Taken together, these results suggest that cotreatment with EBB and doxorubicin could strongly potentiate the antitumor activity of doxorubicin, thus may have significant clinical application in cancer chemotherapy.